Volume 17, Number 3, September 2005

Suitability of DNA Extracts from Peripheral Blood, Oral Mucosa, Dental Pulp, and Split Tooth for Genetic Analysis

Ah-Hup Michael Ong,1 Jin-Ai Mary Anne Tan2
1Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, and 2Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia


Abstract
Objective: To assess the suitability of DNA extracted from peripheral blood, oral mucosal tissue, dental pulp, and split tooth for genetic analysis.
Materials and Methods: DNA was isolated and extracted from the blood, oral mucosal tissue, dental pulp, and split tooth of 5 patients undergoing surgical removal of an impacted third molar. Six millilitres of peripheral blood was collected in an ethylenediamine tetra-acetic acid tube and frozen immediately. A piece of tissue from the buccal mucosal flap was removed following surgery. The pulp was harvested by cutting the extracted molar in half and scraping with a curette. DNA was extracted from both halves of the tooth. All samples were extracted in Tris-ethylenediamine tetra-acetic acid (pH 8) using sodium dodecyl sulphate and proteinase K, and digested overnight at 37ºC. DNA was purified using phenol-chloroform–isoamyl alcohol, precipitated in 4 M sodium acetate with ethanol, and assessed by amplification of the variable number of tandem repeat locus DIS80 using polymerase chain reaction. The presence of contaminating bacteria in the samples was analysed by detection of the bacterial 16S ribosomal gene.
Results: The DNA purity in 90% of the samples was 1.5 to 1.7, which indicated good purity with minimal protein contamination. Amplification of the extracted DNA at the variable number of tandem repeat locus DIS80 produced distinct bands in DNA extracted from the 4 different samples. Individuals homozygous for the DIS80 locus in chromosome 1 produced equal numbers of repeats from both homologues of chromosome 1. DNA from heterozygous individuals produced 2 bands as a result of amplification of a different number of repeats in the 2 chromosomes.
Conclusions: This preliminary study indicates that pure and amplifiable DNA can be successfully extracted from blood, oral mucosal tissue, dental pulp, and split tooth.

Key words:
Blood, Dental pulp, Gene amplification, Mucous membrane, Tooth

Asian J Oral Maxillofac Surg. 2005;17:162-167.
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